MicroRNAs (miRNAs) are the focus of current biomedical research from the perspective of its role in pathogenesis, diagnosis, and therapeutic intervention of several disease states. But the lack of method that is rapid, quantitative, sensitive, and tolerant to matrix effects imposes limit on measurement of miRNAs in clinical samples and in biomedical research. Our long term goal is to develop rapid, quantitative, and highly sensitive tests for the detection of a panel of miRNA targets in cellular matrix that are vital in biomedical research and disease diagnosis. The objective of this application is to develop highly sensitive one-step detection method for target miRNAs in a cellular extract using split-bioluminescent reporter enzyme followed by demonstrating the application of the strategy in fundamental biomedical research. The central hypothesis of the application is that the formation of an active enzyme from the split enzyme fragments conjugated to oligonucleotide probes upon hybridization provides for the development of mix-and-measure assay, and the use of bioluminescent enzyme allows for direct detection in a cellular extract because there is no background signal associated with bioluminescence signal detection. The rationale for this project is that its successful completion is expected to provide a highly sensitive, quantitative, and rapid one-step method for the detection of any desired miRNA target. Thus the proposed research is relevant to the NIH's mission that pertains to developing sensing of biomolecules that perform essential function in human health. Our hypothesis will be tested by pursuing two specific aims, 1) Design and development of sensitive, mix-and-measure assay method for miR21, miR493, and miR511, 2) Determination of alteration in the levels of target miRNAs in a cellular extract of MCF-7 breast cancer cells exposed to estrogen. The proposed work is innovative, because it introduces a new approach for the detection of microRNA in complex matrix such as cellular extract capitalizing on the use of highly sensitive bioluminescent detection system. The proposed research is significant, because it is expected to provide a sensitive and quantitative tool for the measurement of miRNA that will have an impact on the diagnostics and research performed toward understanding the role of miRNA in human health. [unreadable] MicroRNAs (miRNA) have been found to regulate multiple genes associated with diverse cellular functions such as apoptosis, cell differentiation, fat metabolism, and early development. The role of miRNA in the disease progression is now being heavily investigated from the perspective of pathogenesis, diagnosis, and disease prevention. In that regard, the proposed work is important because it will provide tools for such investigations to be performed in a rapid and sensitive manner. The proposed research is expected to provide an excellent research and diagnostic tool that is essential in investigating the impact of microRNAs on human health. [unreadable] [unreadable] [unreadable]